Optically active pyridobenzoxazine derivatives and intermediates thereof

ABSTRACT

An optically action pyridobenzoxazine derivative, a process for preparing the same and a novel intermediate useful for preparing the optically active pyridobenzoxazine derivative are disclosed. The optically active pyridobenzoxazine derivative possesses increased antimicrobial activity and reduced toxicity. The intermediate is useful for preparing such optically active pyridobenzoxazine derivatives such as Ofloxacin and anolog compounds.

This is a continuation of application Ser. No. 07/559,033 filed Jul. 30,1990, now abandoned in turn a continuation of U.S. application No.07/327,653 filed Mar. 23, 1989, now U.S. Pat. No. 4,985,557 in turn adivisional application of application No. 06/876,623 filed Jun. 20,1986, now U.S. Pat. No. 5,053,407.

FIELD OF THE INVENTION

This invention relates to optically active pyridobenzoxazine derivativesand a process for preparing the same and to novel intermediates usefulfor preparing such derivatives. More particularly, it relates tooptically active compounds of Ofloxacin and its analogs, a process forpreparing the same and intermediates useful for preparing the same.

BACKGROUND OF THE INVENTION

Ofloxacin((±)-9-fluoro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de][1,4]-benzoxazine-6-carboxylicacid) is known to be an excellent synthetic antimicrobial agent asdisclosed in Japanese Patent Application (OPI) No. 46986/82 (the term"OPI" used herein means "unexamined published patent application").

Ofloxacin has an asymmetric carbon atom at the 3-position thereof and isobtained as a racemate (specific rotation [α]_(D) =0°) by knownprocesses. The present inventors obtained optically active compounds ofthe racemic Ofloxacin and found that the S(-)-compound possesses anantimicrobial activity of about 2 times higher than that of the(±)-compound and an acute toxicity (LD₅₀) weaker than that of the(=)-compound as determined in mice by intravenous administration. On theother hand, the present inventors have found that the R(+)-compoundexhibits an antimicrobial activity of only about 1/10 to 1/100 timesthat of the (±)-compound, whereas it possesses an acute toxicitysubstantially equal to that of the (±)-compound. That is, the S(-)-formof Ofloxacin has been found to have very desirable properties, i.e.,increased antimicrobial activity and reduced toxicity, and is expectedto be a very useful pharmaceutical agents as compared with the(±)-compound. Further, both the (R+)- and S(-)-compounds of Ofloxacin inthe free form have markedly high water-solubility as compared with(±)-compound and as compared with free compounds of this type, and canbe used as injectable preparations. These advantages will be apparentfrom the experimental data shown hereinafter.

SUMMARY OF THE INVENTION

As a result of investigations with the purpose of preparing, especiallyS(-)-form having higher activity, among the two isomers of Ofloxacin, ithas now been found that compounds having the following formula (X) areuseful as intermediates for synthesizing an isomer of Ofloxacin as wellas other isomers of pyridobenzoxazine derivatives having excellentantimicrobial activity: ##STR1## wherein X₁ and X₂, which may be thesame or different, each represents a halogen atom, such as a fluorineatom, a chlorine atom, and preferably both X₁ and X₂ are fluorine atoms;R₁ represents an alkyl groups having from 1 to 4 carbon atoms, such as amethyl group, an ethyl group, a propyl group, etc., and preferably amethyl group; Q represents a hydrogen atom or a group ##STR2## whereinR₂ represents a substituted sulfonyl group, an alkoxycarbonyl group oran aralkyloxycarbonyl group, such as a p-toluenesulfonyl group, abenzenesulfony group, a methanesulfonyl group, a t-butoxycarbonyl group,a benzyloxycarbonyl group, a p-methoxybenzyloxycarbonyl group, etc.,preferably a substituted sulfonyl group, and more preferably ap-toluenesulfonyl group; and n represents an integer of from 1 to 3, andpreferably 1 or 2.

An object of this invention is to provide optically active Ofloxacin andits analogs.

Another object of this invention is to provide a novel intermediaterepresented by the above-described formula (X) which is useful forsynthesizing optically active Ofloxacin and other pyridobenzoxazinederivatives.

A still another object of this invention is to provide a novel processfor preparing optically active Ofloxacin and its analogs by the use ofthe above-described intermediate.

DETAILED DESCRIPTION OF THE INVENTION

The optically active Ofloxacin and its analogs according to the presentinvention can be represented by the formula (IV): ##STR3## where X₁ andR₁ are as defined above, and R₃ represents an alkyl group having 1 to 3carbon atoms.

In the above-described formula (IV), X₁ preferably represents a fluorineatom, and R₁ preferably represents a methyl group, and R₃ preferablyrepresents a methyl group or an ethyl group.

The optically active Ofloxacin and its analogs of the invention can beprepared by any of Processes A, B and C shown below: ##STR4## whereinX₁, X₂ and R₃ are as defined above.

More specifically, the (±)-3-hydroxymethyl compound (I) is treated with3,5-dinitrobenzoyl chloride, etc. to obtain its derivative, such as the(±)-3,5-dinitrobenzoate compound (II), which is then optically resolvedinto two optically active compounds by an appropriate method, such ashigh performance liquid chromatography (HPLC). The subsequent procedurescan be applied to either of the S(-)-compound and the R(+)-compound tolead to the respective final product, but in view of the purpose of thepresent invention, the final product (VI') is shown as S(-)-form.

The resulting optically active compound is treated with sodiumhydrogencarbonate, etc. to selectively hydrolyzing the benzoate moietyto form the hydromethyl compound (III). The hydroxymethyl compound isconverted into the 3-iodomethyl compound (IV) by using a iodinatingreagent, which is then reduced with n-tributyltin hydride, etc. toprepare the 3-methyl compound. This compound, without being isolated andpurified, can be hydrolyzed as such under an acidic condition to obtainthe 3-methyl-6-carboxylic acid (V'). The carboxylic acid is then reactedwith an N-alkylpiperazine by, for example, heating while stirring toobtain the 10-(4-alkylpiperazinyl) compound (VI') as a final product.

The compounds of the formula (VI) wherein R₁ represents an alkyl groupother than the methyl group can also be prepared in the same manner froman appropriate (±)-3-hydroxyalkyl compound of the formula (I). ##STR5##wherein X₁, X₂ and R₃ are as defined above.

As described previously,7,8-difluoro-2,3-dihydro-3-methyl-4H-[1,4]benzoxazine having the formula(X) wherein X₁ and X₂ represents fluorine atoms, Q represents a hydrogenatom and R₁ represents a methyl group is an important intermediate forsynthesizing Ofloxacin. The inventors have conducted variousinvestigations on an advantageous process for preparing an opticalisomer of this compound expecting that such would be a useful startingmaterial for synthesizing the S(-)-isomer of Ofloxacin.

As a result, it has been found that when a racemic7,8-dihalogeno-2,3-dihydro-3-acetoxymethyl-4H-[1,4]benzoxazine (VII), asa substrate, is hydrolyzed with an appropriate enzyme, such as a certainkind of lipase, to form a7,8-dihalogeno-2,3-dihydro-3-hydroxmethyl-4H-[1,4]benzoxazine (VIII),there is a difference in the rate of hydrolysis between the (+)-isomerand the (-)-isomer.

For example, the compound (VII) was reacted with lipoprotein lipase (LPLAmano 3 derived from Pseudomonas aeruginosa, produced by Amano SeiyakuK.K.) or lipase (derived from Porcine pancreas, produced by ShigmaChemical Company (U.S.A.); derived from Canadian cylindracea, producedby Shigma Chemical Company; or derived from Rhizopus delemar, producedby Seikagaku Kogyo Co., Ltd.), and the reaction change with time wasdetermined through HPLC (column: TSk gel ODS-120A, 4.6×250 mm; solvent:acetonitrile/water=1/1 by volume; velocity: 1 ml/min). When the rate ofhydrolysis reached about 55%, the compound (VII) was recovered and ledto a 3,5-dinitrobenzoyl derivative (IX). The resulting reaction productwas quantitatively determined by HPLC (column: Sumipacks OA-4200,4.0×250 mm; solvent: n-hexane/1,2-dichloroethane/ethanol=92/6.4/1.6 byvolume; velocity: 1.6 ml/min) to obtain a ratio of the(+)-isomer/(-)-isomer of the compound (VII). The results obtained wereshown in Table 1 below.

                  TABLE 1                                                         ______________________________________                                                        Rate of                                                       Enzyme          Hydrolysis                                                    (Origin)        (%)       (+)/(-)                                             ______________________________________                                        LPL Amano 3     54.7      23.0/77.0                                           (P. aeruginosa)                                                               Lipase          53.6      42.6/57.4                                           (R. delemar)                                                                  Lipase          54.5      61.9/38.1                                           (C. cylindracea)                                                              Lipase          55.2      56.8/43.2                                           (P. pancreas)                                                                 ______________________________________                                    

These results lead to a conclusion that the optically active compounds(VII) and (VIII) can be obtained by utilizing the so-called asymmetrichydrolysis with these enzymes.

Process B according to the present invention comprises reacting aracemic 7,8-dihalogeno-2,3-dihydro-3-acetoxymethyl-4H-[1,4]benzoxazine(VII) with an assymetric hydrolytic enzyme to recover a mixturecomprising the starting compound (VIII) rich in either one of opticalisomers and a 3-hydroxymethyl compound (VIII), separating the mixtureinto each compound, dinitrobenzoylating either one or both of thesecompounds and further acetylating the benzoylated 3-hydroxymethylcompound (VIII) to obtain the compound (IX), separating the compoundinto a racemate and an optical isomer by crystallization, subjecting theresulting optical isomer to deacylation and dehydroxylation by means ofknown chemical processes to obtain an optically active7,8-dihalogeno-2,3-dihydro-3-methyl-4H-[1,4]benzoxazine (X') and thenobtaining therefrom optically active Ofloxacin or its analog (VI') byknown processes.

Process B of the invention will further be illustrated in some detailreferring to specific examples.

The racemic compound (VII) is dissolved in a 0.1M phosphoric acid buffer(pH 7.0), and liproprotein lipase (LPL Amano 3) is added to the solutionto cause enzymatic reaction at 37° C. By this reaction, the (+)-compoundis preferentially hydrolyzed to thereby form a mixture of the compound(VII) rich in the (-)-isomer and the compound (VIII) rich in the(+-isomer. The reaction mixture is recovered in an appropriate stage byextraction with an organic solvent, such as ethyl acetate.

The above enzymatic reaction may also be carried out in an appropriateorganic solvent, such as a mixed solvent of benzene and n-hexane, byusing a hydrophilic resin, e.g., DEAE-Toyopearl 650M or Toyopearl HW-40,etc., or Celite as a dispersing agent or by using a resin, such asAmberlite XAD-7, Butyl-Toyopearl 650M, etc., as an adsorptive fixingagent. In addition, use of inclusive fixing agents, such asphoto-crosslinked resins, urethane prepolymers, etc., is also consideredto make it possible to effect the enzymatic reaction in an organicsolvent.

The reaction is an organic solvent with an aid of appropriate dispersingagents or fixing agents as described above is advantageous in that thesubstrate can be reacted at higher concentrations than in aqueoussolution and that post-treatments after the reaction can be simplified.Actually, in the case of performing the reaction in an organic solvent,the reaction mixture can be recovered in high yields simply by filteringthe dispersing agent or fixing agent in an appropriate stage andconcentrating the filtrate. Moreover, the fixing agents can berepeatedly reused to advantage.

The compounds (VII) and (VIII) in the reaction mixture can be separatedand purified by a usual method of separation, such as silica gel columnchromatography. The thus separated compound (VII) is treated, forexample, with 3,5-dinitrobenzoyl chloride in tetrahydrofuran in thepresence of pyridine to form the 3,5-dinitrobenzoyl derivative (IX),which is then recrystallized from an appropriate solvent, e.g., a mixedsolvent of ethyl acetate and n-hexane, whereby the racemate ispreferentially crystallized. The racemic crystals are separated byfiltration, and the(-)-7,8-dihalogeno-2,3-dihydro-3-acetoxymethyl-4H-[1,4]benzoxazine3,5-dinitrobenzoyl derivative (IX) having a high optical purity isobtained from the filtrate.

The compound (IX) is then subjected to de-acylation treatment by, forexample, hydrolysis under an alkaline condition to form the (-)-isomerof the compound (VIII). This compound is dissolved in pyridine andtreated with thionyl chloride, and the product is further subjected todehydroxylation in a usual manner, such as reduction with sodiumborohydride in dimethyl sulfoxide, to thereby obtain the(-)-7,8-dihalogeno-2,3-dihydro-3-methyl-4H-[1,4]benzoxazine (X') havingan optical purity of 99% or higher.

When the racemic compound (VII) is treated with lipase (derived fromCandida cylindracea or Porcine Pancreas), the (-)-compound ispreferentially hydrolyzed to obtain the compound (VIII) rich in the(-)-isomer. The resulting compound is led to a compound in the form ofthe compound (IX), which is then treated in accordance with theprocedures as described above, such as separation by crystallization, toform the (-)-isomer of the compound (VIII). the (-)-isomer of thecompound (X') can then be prepared from this product in the same manneras described above with a high purity.

Other asymmetric hydrolyses which can achieve the object of the presentinvention in addition to the above-recited enzymes can be found based onthe above-mentioned elucidation. Further, when it is intended to obtain(+)-compounds, the same procedures as described above can be followedbased on the above elucidation.

The (-)-compound of Ofloxacin and analogs thereof can be prepared fromthe novel intermediates of the present invention having the formula (X')in accordance with Process C as illustrated below. ##STR6## wherein n,X₁, X₂, R₁, R₂ and R₃ are as defined above, and X₃ is a carboxyl groupor a reactive derivative thereof, for example, an active ester, a halideor an acid anhydride of the carboxylic acid.

In Process C, the 7,8-dihalogeno-1,4-benzoxazine derivative (X') iscondensed with a cyclic amino acid or a reactive derivative thereof(XIV) through amide linkage formation to produce the compound (X"). Thecondensation reaction can be effected by either one of the active estermethod, the acid anhydride method or the DCC method, but, generally, thecompound (X') and an acid chloride (XIV) wherein X₃ is --COCl arereacted in an organic solvent such as halogenated hydrocarbons, e.g.,dichloromethane, in the presence of an acid acceptor such as pyridine,triethylamine or potassium carbonate, at room temperature whilestirring. The reaction product can be isolated and purified in aconventional manner, such as crystallization, column chromatography,etc.

In this condensation reaction, when either one of the two isomers of thecyclic amino acid or its reactive derivative (XIV), i.e., an S-compoundor an R-compound, is used, separation of the diastereomeric mixture ofthe compound (X") can be carried out easily. More specifically,derivatives (XIV), such as S- or R-proline, S- or R-pipecolic acid(piperidine-2-carboxylic acid), etc., are suitably used. The mostpreferred compounds (XIV) include (S)-N-benzenesulfonylproline and(S)-N-p-toluenesulfonylproline.

The diasteromeric mixture of the compound (X") can be separated byfractional crystallization, chromatography using silica gel, etc. as acarrier, or a combination thereof.

The thus separated diastereomer is hydrolyzed, usually under a basiccondition, to form a 7,8-diahlogeno-3-(S or R)-loweralkyl-[1,4]benzoxazine (X). This compound can be led to a9,10-dihalogeno-3-(S or R)-lower alkyl7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid(V) through known reactions, which can then be converted to a10-(4-alkylpiperazinyl) compound (VI).

Of the above-described Processes A, B and C, the particularly preferredis Process C.

ANTIMICROBIAL ACTIVITY

The antimicrobial activities of the optical isomers [(-) and (+)] ofOfloxacin and an analog compound to various microorganisms were comparedwith that of Ofloxacin (racemate), and the results are shown in Table 2below. The test method was in accordance with the standard methodspecified by The Japan Society of Chemotherapy.

                                      TABLE 2                                     __________________________________________________________________________    Minimum Inhibitory Concentration (MIC; μg/ml)                                           S(-)- Racemic                                                                              R(+)- Compound of                                   Microorganism                                                                              Ofloxacin*                                                                          Ofloxacin                                                                            Ofloxacin                                                                           Example 17                                    __________________________________________________________________________    E. coli, NIHJ                                                                              <0.1  <0.10  0.39  <0.1                                          K. pneumoniae, Type 2                                                                      <0.1  0.10   1.56  <0.1                                          Ser. marcescens, 10100                                                                     <0.1  0.10   1.56  <0.1                                          Ps. aeruginosa, 32104                                                                      0.39  0.78   12.5  0.39                                          Ps. aeruginosa, 32121                                                                      0.10  0.20   6.25  0.10                                          Ps. maltophilia, IID1275                                                                   0.39  0.78   12.5  0.10                                          S. aureaus, Smith                                                                          0.10  0.20   6.25  <0.1                                          S. aureaus, 209P                                                                           0.20  0.39   25    0.10                                          Str. pyogenes, G-36                                                                        0.78  1.56   <100  1.56                                          __________________________________________________________________________

ACUTE TOXICITY

The acute intravenous toxicity of (±), R(+) and S(-)-forms of Ofloxacinin male mice is shown in Table 3 below.

                  TABLE 3                                                         ______________________________________                                                          Day after                                                            Dose     Numbers   treatment                                         Compounds                                                                              (mg/kg)  of mice   1   2    3   Mortality                            ______________________________________                                        (±)   100      5         0   0    0   0/5                                           200      5         2   0    0   2/5                                           400      5         5   0    0   5/5                                  R(+).sup.                                                                              100      5         0   0    0   0/5                                           200      5         3   0    0   3/5                                           400      5         5   0    0   5/5                                  S(-).sup.                                                                              100      5         0   0    0   0/5                                           200      5         0   0    0   0/5                                           400      5         5   0    0   5/5                                  ______________________________________                                         LD.sub.50 (i.v. in mice)                                                      (±)form 203 mg/kg                                                          S(-)form 244 mg/kg                                                       

SOLUBILITY

The solubility of (±), R(+) and S(-)-forms of Ofloxacin in water at atemperature in the range of from 23° to 26° C. is shown in Table 4below.

                  TABLE 4                                                         ______________________________________                                        Compounds   Water-Solubility (μg/ml)                                       ______________________________________                                        (±)       2,400                                                            R(+).sup.   25,800                                                            S(-).sup.   22,500                                                            ______________________________________                                    

The conversion of the intermediates of the formula (X) into the desiredOfloxacin or an analog thereof can be carried out by a well knownprocess as disclosed, for example, in U.S. Pat. No. 4,382,892, EPCPatent 47005, Japanese Patent Application (OPI) Nos. 29789/83 and43977/83.

This invention will now be illustrated in greater detail with referenceto the following examples, but it should be understood that they are notintended to limit the present invention. Examples 1 to 7 describepreparation of Ofloxacin according to Process A; Examples 8 to 11describe preparation of Ofloxacin according to Process B; and Examples12 to 17 describe preparation of Ofloxacin and analog compoundsaccording to Process C.

EXAMPLE 1 Preparation of Benzoyloxy Compound

One gram of(±)-9,10-difluoro-3-hydroxymethyl-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicacid ethyl ester (I) and 500 mg of pyridine were suspended in 100 ml ofanhydrous tetrahydrofuran (THF), and 1.6 g of 3,5-dinitrobenzoylchloride was added thereto, followed by refluxing at 90° C. Thesuspension was once dissolved, and a colorless precipitate was thenformed. The reaction was continued for 1.5 hours. After cooling, theprecipitate was collected by filtration, washed with methanol anddiethyl ether and dried to obtain 1.2 g of(±)-9,10-difluoro-3-(3,5-dinitrobenzoyloxy)methyl-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicacid ethyl ester (II) as a colorless powder having a melting point of240°-242° C.

NMR (CDCl₃ /5% DMSO-d₆) δ(ppm); 1,30 (3H, t, J=7.0 Hz, --CH₂ CH₃), 4.26(2H, q, J=7.0 Hz, --CH₂ CH₃), 4.4-4.5 (5H, m), 7.76 (1H, dd, J=11.0 Hz,7.0 Hz, C₈ --H), 8.8 (1H, s, C₅ --H), 9.0 (2H, d, J=3.0 Hz, aromaticring proton) and 9.2 (1H, t, J=3.0 Hz, aromatic ring proton)

EXAMPLE 2 Optical Resolution

Six milligrams of the benzoyloxy compound as obtained in Example 1 wasdissolved in about 0.6 ml of dimethylformamide (DMF) which had beenpurified by distillation. The solution was filtered through a milliporefilter and subjected to HPLC using a column of Sumipacks OA-4200 (2cm×25 cm) and a solvent of n-hexane/1,2-dichloroethane/ethanol=6/3/1 (byvolume) at a velocity of 8 ml/min.

Since the initial fractions (fractions of the (+)-compound) contained aslight amount of the racemic compound (I) due to partial hydrolysis whendissolved in DMF, they were further purified by silica gelchromatography using chloroform to 10% methanol/chloroform as an eluent.These purification procedures were repeated to thereby obtain 250 mgeach of the two optically active compounds [(-)-isomer and (+)-isomer]from 600 mg of the benzoyloxy compound (II).

(+)-isomer: retention time: 56-76 mins. (column temperature: 22° C.);melting points: 235°-240° C.; [α]_(D) ²³ =+90.8° (c=0.852, DMF)

(-)-isomer: retention time: 78-98 mins. (column temperature: 224° C.);melting points: 244°-249° C.;

[α]_(D) ²³ =-92.5° (c=0.889, DMF)

EXAMPLE 3 Preparation of Ethyl(-)-9,10-Difluoro-3-Hydroxymethyl-7-Oxo-2,3-Dihydro-7H-Pyrido[1,2,3-de][1,4]Benzoxazine-6-Carboxylate(III)

In a mixture of 10 ml of ethanol and 4 ml of a saturated aqueoussolution of sodium bicarbonate, 120 mg of the optically activebenzoyloxy compound [(-)-isomer] was suspended, and the suspension washeated at 50° to 60° C. for 2 hours while stirring. After concentration,water was added to the reaction mixture, and any insoluble material wascollected by filtration, washed successively with water, 95% ethanol anddiethyl ether to obtain 68 mg of an optically active 3-hydroxymethylcompound [(III), (-)-isomer] as a colorless crystal having a meltingpoint of 235°-240° C.

Elementary Analysis for C₁₅ H₁₃ F₂ NO₅ :

Calcd. (%): C 55.39, H 4.03, N 4.31

Found (%): C 55.44, H 4.01, N 4.49 [α]_(D) ²³ =-125.9° (c=0.918, DMF)

In the same manner as described above, a (+)-3-hydroxymethyl compoundwas synthesized from the (+)-benzoyloxy compound. Melting point:231°-234° C. [α]_(D) ²³ =+125.9° (c=0.715, DMF).

EXAMPLE 4 Preparation of Ethyl(-)-9,10-Difluoro-3-Iodomethyl-7-oxo-2,3-Dihydro-7H-Pyrido[1,2,3-de][1,4]Benzoxazine-6-Carboxylate(IV)

In 12 ml of anhydrous DMF was suspended 63 mg of the (-)-3-hydroxymethylcompound (III), and the suspension was heated at 70° to 80° C. withstirring to form a solution, followed by allowing to cool to roomtemperature. To the solution as added 340 mg of triphenylphosphitemethiodide, followed by stirring for 1.5 hours. The solvent was removedby distillation under reduced pressure, and the residue was dissolved inchloroform. The solution was partitioned with a sodium thiosulfateaqueous solution and then with a saturated sodium chloride aqueoussolution. The chloroform layer was dried over anhydrous magnesiumsulfate, and the solvent was removed by distillation. To the residue wasadded diethyl ether, followed by stirring, and the precipitated solidwas collected by filtration, washed with diethyl ether and dried underreduced pressure to obtain 78 mg of an iodomethyl compound (IV) as awhite powder having a melting point of 214°-217° C.

Elementary Analysis for C₁₅ H₁₂ F₂ INO₄ :

Calcd. (%): C 41.40, H 2.78, N 3.22

Found (%): C 41.16, H 2.58, N 2.99

The (+)-compound was obtained in the same manner as described above.

EXAMPLE 5 Preparation ofS(-)-9,10-Difluoro-3-Methyl-7-Oxo-2,3-Dihydro-7H-Pyrido-[1,2,3-de][1,4]-Benzoxazine-6-CarboxylicAcid (V')

In 18 ml of absolute ethanol was suspended 78 mg of the iodomethylcompound (IV), and the suspension was heated at 60° to 70° C. withstirring to form a solution , followed by allowing to cool to roomtemperature. To the resulting solution was added 0.2 ml of n-tributyltinhydride, and the mixture was stirred at 50° to 60° C. for 1 hour andthen at room temperature for 1 hour. The solvent was removed bydistillation, and the residue was subjected to column chromatographyusing 8 g of silica gel as a carrier and chloroform:methanol (40:1 byvolume) as an eluent to obtain a crude methyl compound. The crudeproduct was dissolved in 2 ml of glacial acetic acid, and 4 ml ofconcentrated hydrochloric acid was added thereto. After heating atreflux for 40 minutes, the reaction mixture was concentrated. Water wasadded to the concentrate, and the thus precipitated crystal wascollected by filtration, washed successively with water, ethanol anddiethyl ether and dried under reduced pressure to obtain 22 mg ofcrystals of a S(-)-compound (V') having a melting point of 300° C. orhigher.

Elementary Analysis for C₁₃ H₉ F₂ NO₄ :

Calcd. (%): C 55.52, H 3.23, N 4.98

Found (%): C 55.79, H 3.20, N 4.91

[α]_(D) ²³ =-65.6° (c=0.950, DMSO)

EXAMPLE 6 Preparation ofS(-)-9-Fluoro-3-Methyl-10-(4-Methyl-1-Piperazinyl)-7-Oxo-2,3-Dihydro-7H-Pyrido[1,2,3-de][1,4]-Benzoxazine-6-CarboxylicAcid

In 3 ml of anhydrous dimethyl sulfoxide was dissolved 21 mg of theS(-)-9,10-difluoro-3-methyl-6-carboxylic acid (V') and 30 mg ofN-methylpiperazine, and the solution was stirred at 130° to 140° C. for1 hour. The solvent was removed by distillation, and to the residue wasadded 2 ml of ethanol. The thus precipitated solid was collected byfiltration and washed successively with a small amount of ethanol anddiethyl ether. The resulting powder weighing 14 mg was passed through acolumn of 5 g of silica gel and eluted with a lower layer solution ofchloroform-methanol-water (7:3:1 by volume) to obtain the titledcompound, 10-(4-methyl-1-piperazinyl) compound (VI'). The mother liquorleft after the filtration was subjected to thin layer chromatography(silica gel; 20 cm×20 cm, 5 mm (t)) and developed with a lower layersolution of chloroform-methanol-water (15:3:1 by volume). Both thepurified products were combined to yield 14 mg of the titled compound asa crystal having a melting point of 220°-228° C. (with decomposition).

Elementary Analysis for C₁₈ H₂₀ FN₃ O₄ :

Calcd (%): C 59.82, H 5.58, N 11.63

Found (%): C 60.01, H 5.69, N 11.53

[α]_(D) ²⁴ =-68.8° (c=0.711, 0.05N NaOH)

MS (m/e): 361 (M⁺)

NMR (CDCl₃) δ(ppm): 1.63 (3H, d, C₃ --CH₃), 2.38 (3H, s, N--CH₃),2.54-2.60 (4H, m, 2×CH₂ N), 3.40-3.44 (4H, m, 2×CH₂ H), 4.35-4.52 (3H,m, CH and CH₂), 7.76 (1H, d, aromatic ring C₈ --H) and 8.64 (1H, s, C₅--H)

The (+)-compound was obtained in the same manner as described above.Melting point: 218°-226° C. (with decomposition). [α]_(D) ²⁴ =+68.7°(c=0.560, 0.05N NaOH) MS (m/e): 361 (M⁺).

EXAMPLE 7 Preparation ofS(-)-9-Fluoro-3-Methyl-10-(4-Methyl-1-Piperazinyl)-7-Oxo-2,3-Dihydro-7H-Pyrido[1,2,3-de][1,4]Benzoxazine-6-CarboxylicAcid (VI')

In 30 ml of diethyl ether was suspended 281 mg of the(-)-9,10-difluoro-3-methyl-6-carboxylic acid (V') as obtained in Example5, and a large excess of boron trifluoride ethyl etherate was addedthereto while stirring at room temperature, followed by allowing themixture to react for 45 minutes. The precipitate formed was collected byfiltration, washed with diethyl ether and dried under reduced pressure.The resulting chelate compound weighing 310 mg was dissolved in 6 ml ofdimethyl sulfoxide, and 0.32 ml of triethylamine and 0.13 ml ofN-methylpiperazine were added to the solution. The mixture was stirredat room temperature for 17 hours, followed by concentration to drynessunder reduced pressure. The residue was washed with diethyl ether andthen dissolved in 20 ml of 95% ethanol containing 0.5 ml oftriethylamine, and the solution was heated at reflux for 8 hours. Aftercooling, the reaction mixture was concentrated to dryness under reducedpressure. The residue was dissolved in 5% diluted hydrochloric acid, andthe solution was distributed between chloroform and water. The aqueouslayer was adjusted to a pH of 11 with 1N sodium hydroxide and then to apH of 7.4 with 1N hydrochloric acid. The solution was extracted threetimes with 50 ml portions of chloroform, and the extract was dried oversodium sulfate. The chloroform was removed by distillation.Recrystallization of the resulting powder from ethanol-diethyl ethergave 120 mg of the titled compound as a transparent fine needle-likecrystal having a melting point of 225°-227° C. (with decomposition).[α]_(D) ²⁴ =76.9° (c=0.385, 0.05N NaOH)

Elementary Analysis for C₁₈ H₂₀ FN₃ O₄.1/2H₂ O:

Calcd. (%): C 58.37, H 5.72, N 11.35

Found (%): C 58.17, H 5.58, N 11.27

REFERENCE EXAMPLE 1 Preparation of(±)-3-Acetoxmethyl-7,8-Difluoro-2,3-Dihydro-4H-[1,4]Benzoxazine ##STR7##

In 1.0 liter of acetone was dissolved 60.0 g of2,3-difluoro-6-nitrophenol, and 70.0 g of 1-acetoxy-3-chloro-2-propaneand then 33.1 g of potassium carbonate were added to the solution whilestirring at room temperature. After stirring for an additional 30minutes, 6.6 g of potassium iodide were added thereto, and the mixturewas refluxed for 4 hours. After allowing to cool, the reaction mixturewas filtered, and the filtrate was concentrated under reduced pressure.The concentrate was dissolved in 4.0 liters of a mixed solvent of ethylacetate:benzene (1:1 by volume). The resulting solution was washed withwater, dried over anhydrous sodium sulfate and concentrated underreduced pressure. The concentrate was subjected to column chromatographyusing 1.2 Kg of silica gel and benzene/ethyl acetate (10/1) as an eluentto obtain 32.8 g of Compound (A) as an oily product. Compound (A) wasdissolved in 300 ml of methanol, and 115 ml of Raney nickel was addedthereto to effect catalytic reduction under atmospheric pressure. Thereaction mixture was filtered, and the filtrate was concentrated underreduced pressure. The concentrate was purified by column chromatographyby using 400 g of silica gel and benzene/ethyl acetate (10/1 by volume)as an eluent, and the product was recrystallized from benzene-n-hexaneto obtain 17.9 g of(±)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine (VII)as a colorless crystal having a melting point of 73°-74° C.

Elementary Analysis for C₁₁ H₁₁ F₂ NO₃ :

Calcd. (%): C 54.32, H 4.56, N 5.76

Found (%): C 54.09, H 4.42, N 5.76

EXAMPLE 8 Preparation of(-)-3-Acetoxymethyl-7,8-Difluoro-2,3-Dihydro-4H[1,4]Benzoxazine-3,5-DinitrobenzoylDerivative (IX)

a) Ten grams of(±)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine (VII)as a substrate was dissolved in 1.00 liter of a mixed solvent ofbenzene/n-hexane (4/1 by volume). A resin in a wet state which wasprepared by suspending 100 ml of DEAE-Toyopearl 650M in a 0.05Mphosphoric acid buffer (pH 7.0) followed by filtration by suction and200 mg of lipoprotein lipase (LPL Aman 3) were added to theabove-prepared substrate solution. The reaction system was allowed toreact at 37° C. for 6 hours under stirring. The reaction mixture wasfiltered by suction, and the resin was washed with 200 ml of benzene.The filtrate and the washing were combined and concentrated underreduced pressure. The concentrate weighing 9.68 g was subjected tocolumn chromatography using 200 g silica gel as a carrier andbenzene/ethyl acetate (10/1 by volume) as an eluent to obtain 4.67 g of3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine.

The resulting compound was dissolved in 200 ml of tetrahydrofuran, and5.76 g of 3,5-dinitrobenzoyl chloride and 3.3 ml of pyridine were addedthereto, followed by heating at 60° C. for 3 hours. The reaction mixturewas concentrated under reduced pressure, and the concentrate wasdissolved in 400 ml of ethyl acetate, washed successively with dilutedhydrochloric acid, an aqueous solution of sodium bicarbonate and water,dried over anhydrous sodium sulfate and concentrated under reducedpressure. Addition of n-hexane to the concentrate caused precipitationof pale yellow crystals or a racemate. After sufficient precipitation,the precipitate was separated by filtration, and the filtrate wasconcentrated by dryness to obtain 3.93 g of a 3,5-dinitrobenzoylderivative of the(-)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H[1,4]benzoxazine (IX).

b) To about 2.0 ml of Amberlite XAD 7 was added 2.0 ml of a 0.05Mphosphoric acid buffer (pH 7.0) having dissolved therein 20 mg oflipoprotein lipase (LPL Amano 3), and the system was allowed to stand atroom temperature for 18 hours to thereby adsorb the enzyme onto theresin. The resin was filtered by suction and washed with 10 ml of a0.05M phosphoric acid buffer (pH 7.0). A solution of 250 mg of(±)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine as asubstrate in 25 ml of a mixed solvent of benzene and n-hexane (4:1 byvolume) was added to the thus prepared resin in a wet state, followed byallowing to react at 37° C. for 4 hours under stirring. The reactionmixture was worked-up in the same manner as described in a) above toobtain 117 mg of optically active3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[ 1,4]benzoxazine. In thesame manner, there was obtained 65 mg of a 3,5-dinitrobenzoyl derivativeof the (-)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine.

c) To 3.60 liters of a 0.1M phosphoric acid buffer (pH 7.0) was added3.60 g of(±)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine as asubstrate, and the mixture was stirred at 37° C. for 18 hours to form asolution. To the resulting solution was added 50 mg of liproproteinlipase (LPL Amano 3), followed by allowing the system to react at 37° C.for 190 minutes while stirring. The reaction mixture was extracted threetimes with 2.0 liter portions of ethyl acetate. The combined extract waswashed with water, dried over anhydrous sodium sulfate and concentratedunder reduced pressure. The concentrate was subjected to columnchromatography using 70 g of silica gel as a carrier and chloroform asan eluent to obtain 1.07 g of optically active3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine. Theresulting compound was treated in the same manner as described in a)above to obtain 0.9 g of a 3,5-dinitrobenzoyl derivative of the(-)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine.

d) To 3.70 liters of a 0.1M phosphoric acid buffer (pH 7.0) was added3.70 g of(±)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine as asubstrate, followed by stirring at 37° C. for 3.5 hours to form asolution. To the resulting solution was added 2.22 g of lipase (derivedfrom Candida cylindracea), followed by allowing the system to react at37° C. for 7.5 hours while stirred. The reaction mixture was extractedthree times with 2.0 liter portions of ethyl acetate. The combinedextract was washed with water, dried over anhydrous solution sulfate andconcentrated under reduced pressure. The concentrate was subjected tocolumn chromatography using 70 g of silica gel and developed first withbenzene/ethyl acetate (5/1 by volume) to elute3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine and thenwith benzene/ethyl acetate (1/1 by volume) to elute7,8-difluoro-2,3-dihydro-3-hydroxymethyl-4H-[1,4]benzoxazine.

The later eluate weighing 1.31 g was dissolved in 60 ml oftetrahydrofuran, and 1.70 g of 3,5-dinitrobenzoyl chloride was addedthereto, followed by heating at 37° C. for 20 hours. The reactionmixture was concentrated under reduced pressure, and the concentrate wasdissolved in 400 ml of ethyl acetate, washed successively with a sodiumbicarbonate aqueous solution and water, dried over anhydrous sodiumsulfate and concentrated to dryness sunder reduced pressure to obtain2.52 g of a reaction product. The reaction product was dissolved in 10ml of pyridine, and 10 ml of acetic anhydride was added thereto,followed by heating at 37° C. for 20 hours. The reaction mixture wasworked-up and recrystallized in the same manner as described in a) aboveto remove the racemate crystals to thereby increase optical purity.Since the product still contained slight amounts of the reactionby-products, it was further purified by Toyopearl HW-40-columnchromatography (column: 2.5×95 cm; developing solvent:methanol/acetonitrile=1/1 by volume) and then silica gel columnchromatography (column: 1.8×34 cm; developing solvent;chloroform/acetone=20/1 by volume) to finally obtain 0.44 g of a3,5-dinitrobenzoyl derivative of(-)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine.

MS (m/z): 437 (M⁺)

¹ H-NMR (CDCl₃, 200 MHz) δ(ppm): 2.14 (3H, s, --OCOCH₃), 4.26 (2H, d,J=7.0 Hz, --CH₂ OCOCH₃), 4.45 (1H, dd, J=3.0 Hz, 12.0 Hz, C₂ --H), 4.71(1H, d, J=12.0 Hz, C₂ --e,uns/H/ ), 4.94 (1H, m, C₃ --H), 6.60 (2H, m,aromatic ring proton) 8.73 (2H, d, J=2.0 Hz, aromatic ring proton) and9.19 (1H, t, aromatic ring proton)

EXAMPLE 9 Preparation of(-)-7,8-Difluoro-2,3-Dihydro-3-Hydroxymethyl-4H-[1,4]Benzoxazine (VIII)

In 135 ml of tetrahydrofuran was dissolved 3.03 g of a3,5-dinitrobenzoyl derivative of(-)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine, and135 ml of ethanol and 30 ml of 1.0N potassium hydroxide were added tothe solution. After the reaction mixture was stirred at room temperaturefor 30 minutes, 3 ml of acetic acid was added thereto forneutralization. The mixture was concentrated under reduced pressure, andthe concentrate was dissolved in 400 ml of chloroform, washedsuccessively with a sodium bicarbonate aqueous solution and water, driedover anhydrous sodium sulfate and concentrated to dryness under reducedpressure. The solid was subjected to column chromatography using 40 g ofsilica gel and eluted with chloroform/methanol (50/1 by volume) toobtain 1.17 g of(-)-7,8-difluoro-2,3-dihydro-3-hydroxymethyl-4H-[1,4]benzoxazine.

[α]_(D) ²² =-14.1° (c=1.80, CHCl₃)

¹ H-NMR (CDCl₃, 200 MHz) δ(ppm): 3.5-4.4 (5H, m), 6.30-6.42 (1H, maromatic ring proton) and 6.54-6.74 (1H, m aromatic ring proton)

EXAMPLE 10 Preparation of(-)-7,8-Difluoro-2,3-Dihydro-3-Methyl-4H-[1,4]Benzoxazine (X')

In 20 ml of pyridine was added 1.17 g of(-)-7,8-difluoro-2,3-dihydro-3-hydroxymethyl-4H-[1,4]benzoxazine, and2.77 g of thionyl chloride was added thereto dropwise under ice-cooling,followed by stirring at 50° to 60° C. for 40 minutes. The reactionmixture was concentrated under reduced pressure, and the concentrate wasdissolved in 300 ml of chloroform and washed with 100 ml of a sodiumbicarbonate aqueous solution. The washing was extracted twice with 200ml portions of chloroform. The combined chloroform layers were washedwith water, dried over anhydrous sodium sulfate and concentrated underreduced pressure. The concentrate was subjected to column chromatographyusing 40 g of silica gel and eluted with chloroform to obtain 1.18 g ofthe reaction product as a colorless oil product. This product wasdissolved in 30 ml of dimethyl sulfoxide, and 0.41 g of sodiumborohydride was added thereto, followed by heating at 80° to 90° C. for1 hour. The reaction mixture was dissolved in 500 ml of benzene, washedwith water to remove the dimethyl sulfoxide, dried over anhydrous sodiumsulfate and concentrated under reduced pressure. The concentrate wassubjected to column chromatography using 40 g of silica gel and elutedwith benzene to obtain 0.80 g of(-)-7,8-difluoro-2,3-dihydro-3-methyl-4H-[1,4]benzoxazine as a colorlessoily product.

[α]_(D) ²⁵ =-9.6° (c=2.17, CHCl₃):

¹ H-NMR (CDCl₃ 200 MHz) δ (ppm): 1.20 (3H, d, J=6.0 Hz, --CH₃), 3,53(1H, m, C₃ --H), 3.81 (1H, dd, J=8.0 Hz, 10.0 Hz, C₂ --H), 4.31 (1H, dd,J=3.0 Hz, 10.0 Hz, C₂ --H), 6.24-6.36 (1H, m, aromatic ring proton) and6.52-6.70 (1H, m, aromatic ring proton)

Optical Purity: >99%e.e.

The product was led to the 3,5-dinitrobenzoyl derivative andquantitatively determined by HPLC using a column of Sumipacks OA-4200;4.0×250 mm and a mixed solvent of n-hexane/1,2-dichloroethane/ethanol(90/9.1/0.9 by volume) at a velocity of 1.5 ml/min.

EXAMPLE 11 ##STR8##

To 1.13 g of (-)-7,8-difluoro-2,3-dihydro-3-methyl-4H-[1,4]benzoxazinewas added 1.5 g of diethyl ethoxymethylenemalonate, and the mixture wasstirred at 130° to 140° C. for 70 minutes. The reaction mixture wassubjected as such to column chromatography using 50 g of silica gel andeluted with chloroform to obtain 2.47 g of diethyl[(-)-7,8-difluoro-3-methyl-2,3-dihydro-4H-[1,4]benzoxazin-4-yl]methylenemalonate.This product was dissolved in 5 ml of acetic anhydride, and 10 ml of amixture of acetic anhydride and concentrated sulfuric acid (2/1 byvolume) with stirring under ice-cooling, followed by stirring at 50° to60° C. for 40 minutes. To the reaction mixture were added ice and anaqueous solution of sodium bicarbonate, and the reaction product wasextracted three times with 150 ml portions of chloroform. The combinedextract was washed with water, dried over anhydrous sodium sulfate andconcentrated under reduced pressure. When a solid began to precipitate,a small amount of diethyl ether was added thereto, and the precipitatewas collected by filtration. The precipitate was washed with a smallamount of diethyl ether to yield 1.32 g of (-)-ethyl9,10-difluoro-3-methyl-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylate.

In 12 ml of acetic acid was dissolved 1.20 g of the resulting compound,and 25 ml of concentrated hydrochloric acid was added to the solution,followed by refluxing at 120° to 130° C. for 90 minutes. Upon allowingthe reaction mixture to stand at room temperature, colorless needle-likecrystals were precipitated, which were collected by filtration andwashed successively with a small amount of water, ethanol and diethylether to obtain 0.96 g of(-)-9,10-difluoro-3-methyl-7-oxo-2,3-dihydro-7-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicacid.

In 30 ml of diethyl ether was suspended 324 mg of the resultingcompound, and a large excess of boron trifluoride ethyl etherate wasadded thereto, followed by stirring at room temperature for 30 minutesto form a chelate compound. The product was collected by filtration andwashed with a small amount of diethyl ether to obtain 373 mg of apowder. The powder was dissolved in 7 ml of dimethyl sulfoxide, and 136mg of N-methylpiperazine and 228 mg of triethylamine were added thereto,followed by stirring at room temperature for 17 hours. The reactionmixture was concentrated to dryness under reduced pressure, and to thesolid were added 15 ml of 95% methanol and 0.31 ml of triethylamine. Theresulting mixture was refluxed for 3 hours. The reaction mixture wasconcentrated under reduced pressure, and the residue was filtered andwashed successively with a small amount of ethanol and diethyl ether toobtain 350 mg of a white powder. Recrystallization from a mixed solventof ethanol and thick aqueous ammonia gave 230 mg of S(-)-Ofloxacin.

Melting Point: 225°.227° C. (with decomposition).

[α]_(D) ²³ =-76.9° (c=0.39, 0.05N NaOH)

Ms (m/e): 361 (M⁺)

¹ H-NMR (CDCl₃, 200 MHz) δ (ppm): 1.63 (3H, d, C₃ --CH₃), 2.38 (3H, s,N--CH₃), 2.54-2.60 (4H, m, 2×CH₂ N), 3.40-3.44 (4H, m, 2×CH₂ N),4.35-4.52 (3H, m, CH and CH₂), 7.76 (1H, d, aromatic ring C₈ --H) and8.64 (1H, s, C₅ --H)

EXAMPLE 12 Preparation of3S-(+)-7,8-Difluoro-2,3-Dihydro-3-Methyl-4-[(S)-N-para-toluenesulfonylprolyl]-4H-[1,4]Benzoxazine(X")

A solution of an acid chloride, which was prepared from 61.9 g of(S)-N-toluenesulfonylproline and thionyl chloride, in 350 ml of drieddichloromethane was slowly added dropwise to a solution of 32.8 g of(±)-7,8-difluoro-2,3-dihydro-3-methyl-4H-[1,4]benzoxazine and 28 ml ofpyridine in 300 ml of dried dichloromethane at room temperature understirring. The stirring was further continued for an additional 4 hoursat room temperature. The reaction mixture was washed successively with10% hydrochloric acid, a saturated aqueous solution of sodiumhydrogencarbonate and a saturated aqueous solution of sodium chlorideand dried over anhydrous magnesium sulfate. The dichloromethane wasremoved by distillation, and the oily residue was dissolved in 200 ml ofethyl acetate. To the solution was slowly added dropwise 750 ml ofn-hexane while stirring whereby crystals ((-)-isomer of the compound(X") wherein X₁ =X₂ =F; R₁ =CH₃ : R₂ =p-toluenesulfonyl; and n=1)precipitated immediately. The precipitated crystals were separated byfiltration, and the filtrate was concentrated to dryness under reducedpressure. The residue was subjected to column chromatography using 500 gof silica gel and eluted with benzene/ethyl acetate (50/1-25/1 byvolume) to obtain an oily product. The oily product was dissolved in 500ml of ethanol, and the solution was allowed to stand at room temperaturefor 1 day, thereby to precipitate crystals. The ethanol as distilledoff, and to the thus recover crystals were added diethyl ether andn-hexane, followed by filtration. The solid was dried under reducedpressure to obtain 33.4 g of3S-(+)-7,8-difluoro-2,3-dihydro-3-methyl-4-[(S)-N-p-toluenesulfonylprolyl]-4H-[1,4]benzoxazine((+)-isomer of the compound (X") wherein X₁ =X₂ =F; R₁ =CH₃ ; R₂=p-toluenesulfonyl; and n=1) having a melting point of 107°-108° C.

[α]_(D) =+70.7° (c=0.953, chloroform)

IR ν_(max) ^(KBr) (cm⁻¹): 1685, 1510, 1490

Elementary Analysis for C₂₁ H₂₂ F₂ N₂ O₄ S:

Calcd. (%): C 57.79, H 5.08, N 6.42

Found (%)L C 58.05, H 5.14, N 6.47

EXAMPLE 13 Preparation ofS-(-)-7,8-Difluoro-2,3-Dihydro-3-Methyl-4H-[1,4]Benzoxazine (X)

In 1 liter of ethanol was dissolved 32.8 g of the (+)-isomer as obtainedin Example 12, and 300 ml of 1N sodium hydroxide was added thereto,followed by refluxing for 3 hours. The ethanol was removed bydistillation, and the oily residue was extracted with benzene. Theextract was washed with a saturated aqueous solution of sodium chloride,dried over sodium sulfate and distilled to remove benzene. The residuewas subjected to column chromatography using 200 g of silica gel as acarrier and benzene as an eluent to obtain 12.7 g (yield: 91.4%) ofS-(-)-7,8-difluoro-2,3-dihydro-3-methyl-4H-[1,4]benzoxazine as an oilyproduct.

[α]_(D) =-9.6° (c=2.17, chloroform)

The absolute configuration of this compound was decided to be anS-configuration by X-ray analysis on its hydrochloride.

EXAMPLE 14 Preparation of Ethyl(S)-(-)-7,8-Difluoro-3-Methyl-7-Oxo-2,3-Dihydro-7H-Pyrido[1,2,3-de]-[1,4]Benzoxazine-6-Carboxylate(XII)

To 15.8 g of the (S)-(-)-benzoxazine derivative as obtained in Example13 was added 24.0 g of diethyl ethoxymethylenemalonate, and the mixturewas stirred at 130° to 140° C. for 1 hour under reduced pressure. Aftercooling, the reaction mixture was dissolved in 50 ml of aceticanhydride, and 80 ml of a mixture of acetic anhydride and concentratedsulfuric acid (2:1 by volume) was slowly added dropwise to the solutionwhile stirring under ice-cooling. After continuing the stirring foradditional one hour at room temperature, the reaction mixture wasstirred in a hot bath of 50° to 60° C. for 30 minutes. Ice-water wasadded to the reaction mixture, and the powdery potassium carbonate wasadded thereto for neutralization. The mixture was extracted withchloroform, and the extract was washed successively with a saturatedaqueous solution of sodium hydrogencarbonate and a saturated aqueoussolution of sodium chloride and dried over sodium sulfate. Thechloroform was removed by distillation, and to the residue was addeddiethyl ether. The crystals thus formed were collected by filtration togive 20.0 g of the titled compound having a melting point of 257°-258°C.

[α]_(D) =-68.1° (c=0.250, acetic acid)

EXAMPLE 15 Preparation ofS-(-)-7,8-Difluoro-3-Methyl-7-Oxo-2,3-Dihydro-7H-Pyrido-[1,2,3-de][1,4]Benzoxazine-6-CarboxylicAcid

In 50ml of acetic acid was dissolved 19.5 g of the ester compoundobtained in Example 14, and 400 ml of concentrated hydrochloric acid wasadded thereto, followed by refluxing for 3 hours. After cooling, theprecipitated crystals were collected by filtration, washed successivelywith water, ethanol and diethyl ether and dried to obtain 16.2 g of thecorresponding carboxylic acid having a melting point of 300° C. orhigher.

[α]_(D) =-65.6° (c=0.985, DMSO)

EXAMPLE 16 Preparation ofS-(-)-9-Fluoro-3-Methyl-10-(4-Methyl-1-Piperazinyl)-7-oxo-2,3-Dihydro-7H-Pyrido[1,2,3-de][1,4]Benzoxazine-6-CarboxylicAcid (VI) (S-(-)-Isomer of Ofloxacin)

In 600 ml of diethyl ether was suspended 14.3 g of the carboxylic acidobtained in Example 15, and 70 ml of boron trifluoride diethyl etheratewas added thereto, followed by stirring at room temperature for 5 hours.The supernatant liquid was removed by decantation, and to the residuewas added diethyl ether, followed by filtration. The solid was washedwith diethyl ether and dried. The product was dissolved in 100 ml ofdimethyl sulfoxide, and 14.2 ml of triethylamine and 7.3 ml ofN-methylpiperazine were added to the solution. After the mixture wasstirred at room temperature for 18 hours, the solvent was removed bydistillation. Diethyl ether was added to the residue, followed byfiltration. The collected yellow powder was suspended in 400 ml of 95%methanol, and 25 ml of triethylamine was added thereto. The mixture washeated at reflux for 25 hours. The solvent was distilled off underreduced pressure, and the residue was dissolved in 500 ml of 10%hydrochloric acid and washed three times with chloroform. The washedsolution was adjusted to a pH of 11 with a 4N sodium hydroxide aqueoussolution and then to a pH of 7.3 with 1N hydrochloric acid. The solutionwas extracted three times with 2 liter portions of chloroform, and thecombined extract was dried over sodium sulfate. The chloroform wasremoved by distillation, and the resulting crystal was recrystallizedfrom ethanol/diethyl ether to obtain 12.0 g of the titled compoundhaving a melting point of 226°-230° C. (with decomposition).

[α]_(D) =-76.9° (c=0.655, 0.05N NaOH)

EXAMPLE 17 Preparation of(S)-(-)-9-Fluoro-3-Methyl-10-(4-Ethyl-1-Piperazinyl)-7-Oxo-2,3-Dihydro-7H-Pyrido[1,2,3-de][1,4]Benzoxazine-6-CarboxylicAcid (VI)

In the same manner as described in Example 16 except thatN-ethylpiperazine was used in place of N-methylpiperazine, the titledcompound was obtained having a melting point of 229°-230° C. (withdecomposition).

Elementary Analysis for C₁₉ H₂₂ FN₃ O₄ :

Calcd. (%): C 60.79, H 5.91, N 11.19

Found (%): C 60.97, H 5.91, N 11.30

[α]_(D) =-67.0° (c=0.585, H₂ O)

NMR (CDCl₃) δ (ppm): 1.16 (3H, t, J=7 Hz, --CH₂ CH₃), 1.63 (3H, d, J=7Hz, CH₃), 2.53 (2 H, q, J=7 Hz, CH₂ CH₃), 2.57-2.69 (4H, m, 2×CH₂),3.40-3.53 (4H, m, 2×CH₂), 4.32-4.58 (3H, m, CH and CH₂), 7.77 (1H, d,J=12 Hz, C₈ --H), 8.67 (1H, S, C₅ --H)

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

What is claimed is:
 1. An S(-)-optically pure 4-substituted benzoxazinederivative represented by the formula (X) ##STR9## wherein X₁ and X₂,which may be the same or different, each represents a halogen atom, R₁represents an alkyl group having 1 to 4 carbon atoms, and Q represents ahydrogen atom.